Enzylight

Example 7

3.5×10⁴ MEL-28 cells were plated overnight at 37 degrees C. Cells were washed with Tris assay buffer to remove phosphate. 100 nM titrated down to 0.005 pM of monoclonal antibodies were incubated with cells for 30 minutes at 37 degrees C. 50 μM ATP was added and incubated for 15 minutes. Supernates were harvested and frozen. Supernates were thawed and evaluated for ATP using the EnzyLight (EnzyLight ATP Assay Kit, BioAssay Systems). Palivizumab was used as an isotype control and ARL (Tocris) and POM-1 (Alpha Aesar) used at a concentration of 100 μM are non-specific small molecule inhibitors of CD39 as positive controls.

ATP was almost undetectable after 30 minutes post ATP addition to the cells in untreated and/or isotype treated samples (see FIG. 5 A) while all of the anti-CD39 antibodies tested prevented processing of ATP in dose dependent manner (see FIG. 5 A, B). Most of the anti-CD39 antibodies tested in this assay prevented ATP processing by CD39 to a similar extent as ARL (see FIG. 5A). IC50s of anti-CD39 antibodies in ATP preservation assay ranged from 0.02-0.1 nM. Overall potency of antibodies in this assay was consistent with what was observed in Malachite Green phosphate readout assay.